Method for checking and controlling the mammalian lactic acid fermentation process / aerobic glucose fermentation metabolic pathway in mammalian organism

ABSTRACT

The method for qualitative and quantitative detecting of the extend of use and the correct process flow of the mammalian aerobic glucose fermentation metabolic pathway (mam-aGF) in a mammalian individual is characterized in that the enzyme TKTL1 is used as indicator and target molecule and that the structural and/or functional parameter of said TKTL1 in a biological sample of said individual (patient) are taken as indication for the qualitative and quantitative run of the mam-aGF in the cells and/or tissue of said individual (patient). In combination with the use of inhibitors and activators of that mam-aGF the method is further suitable for checking and controlling the mam-aGF in an individual (patient).

This is the U.S. national stage of International applicationPCT/EP2006/001951, filed Mar. 3, 2006 designating the United States.

The invention relates to (1) a method for qualitative and quantitativedetection of the mammalian lactic acid fermentation process or mammalianaerobic glucose fermentation metabolic pathway, respectively, in amammalian individual (patient, mammalian organism), (2) a method forchecking and controlling (i.e. inhibiting or activating) saidprocess/pathway, and (3) inhibitors and activators of thatprocess/pathway.

The invention is based on the novel scientific discovery, that (1) ametabolism pathway which up to now has been known only from prokaryoticcells (for example from lactobacillus), exists in mammalianorganisms—i.e. in mammalian cells—too, said metabolism pathwaycomprising the energy-yielding breaking down of glucose to lactate,although sufficient free (molecular) oxygen is present and bioavailablei.e. aerobic conditions exist, and that (2) the enzyme TKTL1 is thetracer enzyme of said new glucose fermentation metabolism.

Mammalian cells are able to break down glucose to lactic acid in thecase of oxygen deficiency, finally leading to muscles ache. Thisanaerobic glucose degradation is performed via theEmbden-Meyerhof-pathway. In the 1920s Otto Warburg discovered that alsoin the presence of sufficient available amounts of oxygen mammaliancells are able to break down glucose to lactic acid, i.e. that mammaliancells are able to ferment glucose to lactic acid even in the presence ofsufficient amounts of free bioavailable oxygen. He observed thisanaerobic glucose to lactate degradation pathway in the presence ofoxygen in tumor tissues as well as in certain healthy tissues likeretina and testis. For historical reasons this glucose degradation tolactate even in the presence of oxygen is named aerobic glycolysis orWarburg effect. Since the start and end point (glucose and lactate) isthe same as the glucose fermentation to lactate in the absence of oxygenvia the Embden-Meyerhof pathway, the lactate production has beeninterpreted as the result of the Embden-Meyerhof pathway. However, inthe course of the experiments leading to the present invention it becameobvious that it is the TKTL1 transketolase which allows the glucosedegradation or glucose fermentation, respectively, to lactate even inthe presence of oxygen and that this glucose pathway is significantlydifferent from the Embden-Meyerhof pathway.

The TKTL1 transketolase gene has been discovered some years ago, namely1996 by Coy et al. (Genomics 32, 309-316). Due to a stop codon in apredicted coding exon, the TKTL1 transketolase gene has been annotatedas a pseudogene in sequence data bases. Despite this, in 2002 Coydemonstrated that the TKTL1 gene encodes an enzymatically activetransketolase. The TKTL1 transketolase is phylogentically cognate withthe two other known human transketolases TKT and TKTL2. However, allthree transketolase enzymes are encoded by different genes, whereby thegene for TKT is located on chromosome 3, the gene for TKTL2 is locatedon chromosome 4 and the gene for TKTL1 is located in band Xq28 onchromosome X. in humans.

One most important function of the enzyme TKTL1 in mammalian cells isits catalysator function during the fermentation of glucose to lacticacid in the presence of oxygen, i.e. under aerobic conditions. TKTL1 isthe tracer enzyme in the novel, first recently discovered so calledmammalian aerobic glucose fermentation metabolic pathway or mammalianlactic acid fermentation process, respectively. In the following thisnewly discovered pathway is referred to as mammalian aerobic glucosefermentation metabolic pathway or shortened to mam-aGF, respectively.

Details of that mam-aGF are the following:

A protein complex containing TKTL1 and glyceraldehyde-3-phosphatedehydrogenase (GAPD) allows a nonoxidative glucose degradation. Theelectron transfer does not involve mitochondria and allows amitochondria independent ATP production in TKTL1 expressing cells.Application of anti-TKTL1 compounds, or inhibitory thiamin-analogs, orparabenzoquinons (or benzoquinone-derivatives) can be applied for theinhibition of such a mitochondria independent ATP production (inparticular for example in tumors with a TKTL1 based sugar metabolism).

Using the human TKTL1 protein the inventor of the present patentapplication confirmed the formation of glyceraldehyde-3-phosphate in anone-substrate reaction utilizing X5P as sole carbon source.

The metabolism of X5P via TKTL1 result in the formation of acetyl-CoA.The anaerobic glucose degradation under aerobic conditions leads tolactate and the building of the energy rich compound acetyl-CoA. Thepyruvate dehydrogenase complex is then e.g. inhibited by acetyl-CoA, andas a consequence, pyruvate is mainly reduced to lactate.

A schematic description of the complete mam-aGF are given in FIG. 13 andFIG. 14.

Details concerning the tracer enzyme TKTL1 of that mam-aGF are thefollowing:

During the experiments leading to the present invention different TKTL1isoforms were identified on protein level using a novel monoclonalantibody (Linaris Biologische Produkte GmbH, Wertheim) specificallydetecting the TKTL1 protein.

TKTL1 protein isoforms are part of a multi-protein complex. Within thiscomplex TKTL1 is also bound to transketolase unrelated proteins likeglyceraldehyde-3-phosphate dehydrogenase (GAPDH), DNaseX(DNase1-like-1), Akt (=protein kinase B); histones, histon deacetylase,amyloid precursor protein and actin binding proteins.

Known transketolases are homodimers of two full length proteinsharbouring all typical invariant transketolase amino acid residues. Thetransketolase-like gene encoded TKTL1 protein isoforms build TKTL1homo/heterodimers and TKT/TKTL1 or TKTL2/TKTL1 heterodimers. Theexpression of TKTL1 protein isoforms—even an enzymatically non-activeisoform—influences the enzymatic activity of a TKT or TKTL2 protein aspart of a TKT/TKTL1 or TKTL2/TKTL1 heterodimer. A molecular switch and aproton wire synchronize the active sites in TKT/TKTL1, TKTL2/TKTL1 andTKTL1/TKTL1 homo- and heterodimers.

The proof that the TKTL1 gene (NM_(—)012253; Accession Numbers: X91817;BC025382) encodes a full length transketolase protein as well as smallerprotein isoforms has important implications for basic research andmedical health.

Besides their enzymatic functions, the TKTL1 proteins exhibit variousdifferent functions depending on the localization of the proteins in themammalian cell and on their state of aggregation. In mammalian cells theTKTL1 protein is mainly located in the cytoplasm, but also occurs in thenucleus. Within the cytoplasm the main function of TKTL1 is thecatalysis of the (trans-)ketolase reaction. Additional functions of theTKTL1 proteins located within the nucleus are associated with thecontrol of the cell cycle and mitosis, control of transcription (theTKTL1 gene itself and others), and regulation of apoptosis.

The TKTL1 proteins (with their functions depending on their localizationor the state of aggregation) are designated as “moonlighting” proteins,since they execute different functions depending on subcellularlocalization, the cell type as well as its aggregation state.

The present invention is based on the object (a) of making available amethod for a qualitative and quantitative detecting (and monitoring) ofthe extend (level) of use and the correct (normal, natural) process flowof the mammalian aerobic glucose fermentation metabolic pathway(mam-aGF) in a mammalian individual, i.e. a method for controllingwhether that metabolism/pathway/process actually proceeds in theinvestigated cells of the appropriate mammalian organism, if the case isgiven in what extend and whether it proceeds “normal” or correct,respectively or with faults or aberrations, and (b) of making availablea means with which that mam-aGF can be affected, in particular enhancedor inhibited.

This object is achieved with a (in-vitro-) method for the qualitativeand quantitative detecting (and monitoring) the extend (level) of useand the correct (normal, natural) process flow of the eukaryotic aerobicglucose fermentation metabolic pathway “mam-aGF” (or mammalian lacticacid fermentation process) in a mammalian individual (patient),characterized in that

(a) the enzyme TKTL1 is used as indicator and target molecule and

(b) said method comprises the following steps:

-   -   taking (harvesting, collecting) a biological sample of said        individual (patient),    -   determining the activity and/or concentration, and/or cellular        localization and/or aggregation status and/or dimerization        status of the TKTL1 protein within said sample of said        individual (patient) and within a control sample,    -   comparing the determined data obtained from said sample of said        individual (patient) with the data obtained from the control        sample,    -   and taking (i) an enhanced or decreased level of activity and/or        concentration of the TKTL1 protein in said sample of the        individual compared to the control sample as indication of an        enhanced or decreased, respectively, extend (level) of use of        the mammalian aerobic glucose fermentation metabolic pathway        “mam-aGF”,        and (ii) an abnormal cellular localization and/or an abnormal        aggregation status and/or an abnormal dimerization status of the        TKTL1 protein in said sample of the individual compared to the        control sample as indication of an abnormal (defective,        disturbed, faulty) mammalian aerobic glucose fermentation        metabolic pathway.

The detection (determination) may be carried out in solution or usingreagents fixed to a solid phase. The detection of one or more molecularmarkers, such as polypeptides or nucleic acids, may be performed in asingle reaction mixture or in two or separate reaction mixtures.Alternatively, the detection reactions for several marker molecules may,for example, be performed simultaneously in multi-well reaction vessels.The markers characteristic for the TKTL1 gene products may be detectedusing reagents that specifically recognize these molecules. Thedetection reaction for the marker molecules may comprise one or morereactions with detecting agents either recognizing the initial markermolecules or recognizing the prior molecules used to recognize othermolecules.

The detection reaction further may comprise a reporter reactionindicating the presence or absence and/or the level of the TKTL1 genemarkers. The reporter reaction may be for example a reaction producing acoloured compound, a bioluminescence reaction, a fluorescence reaction,generally a radiation emitting reaction etc. In a preferred embodiment,different marker molecules may be recognized by agents that producedifferent reporter signals, so that the signals referring to markermolecules could be distinguished.

Applicable formats for the detection reaction according to the presentinvention may be, blotting techniques, such as Western-Blot,Southern-blot, and Northern-blot. The blotting techniques are known tothose of ordinary skilled in the art and may be performed for example aselectro-blots, semidry-blots, vacuum-blots or dot-blots. Amplificationreactions may also be applicable for the detection of e.g. nucleic acidmolecules. Furthermore immunological methods for detection of moleculesmay be applied, such as for example immunoprecipitation or immunologicalassays, such as ELISA, RIA, lateral flow assays, immuno-cytochemicalmethods etc.

On the basis of the information obtained with that method a medicalpractitioner is able to draw conclusions concerning the state of healthof the appropriate individual (patient) and to evolve schedules fortherapy if necessary.

Therefore said method is suitable and intended for monitoring(detecting, surveying and observing) the course of diseases, associatedwith an increased or decreased and/or abnormal, i.e.defective/faulty/disturbed mam-aGF.

The determination of TKTL1 activity and/or concentration may comprisedetermining the level of TKTL1 gene products or determining theenzymatic activity of TKTL1 in a sample.

Monitoring may comprise detecting the level of TKTL1 gene products orTKTL1 enzymatic activity in samples taken at different points in timeand determining the changes in said level. According to said changes thecourse of the disease can be followed. The course of the disease may beused to select therapy strategies for the particular individual.

Another aspect of detecting and monitoring of the disease course maycomprise the detection of minimal residual disease. This may comprisefor example the detection of a TKTL1 gene products level or TKTL1enzymatic activity in one or more body samples following initial therapyof an individual once or at several time points. According to the levelof TKTL1 gene products detected in the samples one may select a suitabletherapy for the particular individual.

Based upon the determined level of TKTL1 gene products or the determinedenzymatic activity in the samples individuals can be subdivided intosubgroups. Based upon these subgroups an assessment of prognosis may bedone. According to the subgroups the therapy of the individuals affectedby the various diseases may be tailored. For example the overexpressionof TKTL1 gene and an enhanced activity of the pentose-phosphate cyclesuggest a mechanism by which thiamine (vitamin B1) promotes nucleic acidribose synthesis and an enhanced glucose metabolism. Therefore thethiamine intake has direct consequences for a disease with anoverexpression of the transketolase like-1 gene. This provides alsobackground information and helps to develop guidelines for alternativetreatments with antithiamine transketolase inhibitors in the clinicalsetting. Analysis of RNA ribose indicates that glucose carbonscontribute to over 90% of ribose synthesis in cultured cervix andpancreatic carcinoma cells and that ribose is synthesized primarilythrough the thiamine dependent transketolase pathway (>70%).Antithiamine compounds significantly inhibit nucleic acid synthesis.Thiamine or benfotiamine treatment activates TKTL1 and thereby the sugarmetabolism and reduces toxic or unwanted reactions (e.g. AGE formation,glyoxal.

In addition said method may comprise the detection of auto-antibodiesdirected against polypeptides encoded by the TKTL1 gene. Thepolypeptides used for the methods according to the present invention maybe used to detect the presence or absence of such antibodies in bodyfluids by methods known to those of skill in the art.

Said method is further suitable for performing an in-vivo or in-vitromolecular imaging so that it is possible to identify diseases associatedwith an increased or decreased or abnormal i.e. defective mam-aGF in avery early stage, ideally before the appearance of the typically knownsymptoms of that disease. In consequence the individual or its doctor isable to medicate that disease at a very early point of time therebysignificantly increasing the chances of healing.

Molecular imaging differs from conventional techniques because itidentifies specific gene products and intracellular processes likespecific enzyme reactions. The altered substrate specificity andreaction modus of the TKTL1 enzyme can be used for the detection ofcells or tissues with an enhanced or reduced TKTL1 enzyme activity. Thealtered substrate specificity and reaction modus of the TKTL1 enzymeallows the discrimination between the enzymatic activity of threetransketolase(-like) enzymes thereby allowing the measurement of TKTL1enzymatic activity in vivo. The enzymatic activity can be detected bye.g. positron emission tomography, chemoluminescence or radiographicimaging.

The molecular imaging may be based on the enzymatic conversion of inertor labelled compounds to molecules detectable in the course of molecularimaging methods by the TKTL1 molecules. In another embodiment themolecular imaging method may be based on the use of compounds carrying asuitable label for in vivo molecular imaging, such as radio isotopes,metal ions etc., specifically binding to TKTL1 molecules in vivo.

These compounds are preferably non-toxic and may be eliminated from thecirculation of organisms, such as humans, in a time span, that allowsfor performing the detection of label accumulated in tissueoverexpressing TKTL1 gene. In cases of a molecular imaging, for whichclearance from the circulation is not relevant (for example due to lowbackground produced by the circulating molecules etc.) the compounds foruse should be administered in pharmaceutical acceptable form incompositions that may additionally comprise any other suitablesubstances, such as e.g. other diagnostically useful substances,therapeutically useful substances, carrier substances or the like.

The biological sample of the individual can be almost each tissue orliquid sample obtained from said individual. Isolated cells, lysedcells, cell debris, peptides or nucleic acids of said individual aresuitable samples, too. Further suitable samples are for example biopsypreparations, body fluids, secretions, a smear, serum, urine, semen,stool, bile, a liquid containing cells.

The determination in step (b) of the inventive method can be carried outon the protein level, i.e. with the TKTL1 protein or a TKTL1 proteinfragment as the target. Preferably the determination is carried out byusing a molecule that specifically binds to the TKTL1 protein.

Preferably said molecule is an antibody directed to TKTL1 or a fragmentof such anti-TKTL1-antibody or a peptidomimetic comprising an antigenbinding epitope, or a mini-antibody.

Suitable target molecules are the TKTL1 protein itself or fragmentthereof or a fusion protein comprising the TKTL1 protein.

The determination on the protein level, i.e. of the TKTL1 gene products,can for example be carried out in a reaction comprising an antibodyspecific for the detection of the TKTL1 protein. The antibodies can beused in many different detection techniques for example in Western-blot,ELISA or immunoprecipitation. Generally antibody based detection can becarried out as well in vitro as directly in situ for example in thecourse of an immuno-histochemical staining reaction. Any other methodfor determining the amount of particular polypeptides in biologicalsamples can be used according to the present invention.

The reagent for the detection of the TKTL1 gene product may include anyagent capable of binding to the TKTL1 protein molecule. Such reagentsmay include proteins, polypeptides, nucleic acids, peptide nucleicacids, glycoproteins, proteoglycans, polysaccharides or lipids.

TKTL1 gene products as used in the context of the present invention maycomprise polypeptides and nucleic acids encoded by the transketolaselike-1 gene. The polypeptides and polynucleotides (cf. TKTL1, TKR:NM_(—)012253; Accession numbers: X91817; BC025382) used for performingthe method according to the present invention are isolated. This meansthat the molecules are removed from their original environment.Naturally occurring proteins are isolated if they are separated fromsome or all of the materials, which coexist in the natural environment.Polynucleotides are isolated for example if they are cloned intovectors.

In addition to TKTL1 protein variants with enhanced or reduced levels ofenzymatic activity, TKTL1 proteins with altered localization oraggregation and/or dimerization within the cell can be detected inpatients. Using monoclonal antibodies specifically detecting TKTL1protein (Linaris Biologische Produkte, Wertheim) TKTL1 protein withinthe nucleus can be detected in cells isolated from body fluids. Usingimmunocytochemistry the localization of TKTL1 within the nucleus and thecytoplasm of cells from healthy individuals and patients can bedetermined. In a subset of Alzheimer patients an enhanced localizationof TKTL1 is detectable within the nucleus. A different aggregation ofTKTL1 in Alzheimer patients is also present. Detection of thisaggregation is possible using 2D-gel electrophoresis (FIG. 15). Inaddition an aggregation with other proteins e.g. GAPDH can be detectedby an ELISA using antibodies for TKTL1 and GAPDH.

Just as well the determination in step (b) of the inventive method canbe carried out on the nucleic acid level, i.e. with the TKTL1 nucleicacid or a fragment thereof as the target. The term “TKTL1 nucleic acid”as used in the present context comprises the TKTL1 gene, TKTL1 mRNAs andTKTL1 encoding nucleic acids.

In a preferred embodiment the detection of the above mentioned relevantTKTL1 parameter in the sample should be performed by using at least onenucleic acid probe capable of hybridizing to a TKTL1 nucleic acid. Asuitable target molecule is the TKTL1 nucleic acid itself as well as achimeric nucleic acid comprising a TKTL1 encoding nucleic acid orfragments thereof.

The procedure for the detection of nucleic acids (DNA and/or RNA) can,for example, be carried out by a binding reaction of the molecule to bedetected to complementary nucleic acid probes, proteins with bindingspecificity for the nucleic acids or any other entities specificallyrecognizing and binding to said nucleic acids. This method can beperformed as well in vitro as directly in situ for example in the courseof a detecting staining reaction. Another way of detecting the TKTL1gene products in a sample on the level of nucleic acids performed in themethod according to the present invention is an amplification reactionof nucleic acids, which can be carried out in a quantitative manner suchas for example the polymerase chain reaction. In a preferred embodimentof the present invention real time RT PCR may be used to quantify thelevel of TKTL1 RNA in samples.

The TKTL1 sample for carrying out a positive control may comprise forexample TKTL1 nucleic acids or polypeptides or fragments thereof inapplicable form, such as solution or salt, peptides in applicable form,tissue section samples or positive cells.

In summary: the type (manner) of TKTL1 enzyme activity (normal, reducedor enhanced) can be identified in individuals based on TKTL1 genemutations, reduced or enhanced enzyme activities of the isolated TKTL1protein or an in vivo imaging of the TKTL1 enzyme reaction. Diagnosiscan be performed by determining the enzymatic activity of the TKTL1protein isolated from the patient (e.g. serum, liquor, or other bodyfluids) or by in vivo imaging of TKTL1 enzyme activity.

Kits for performing the inventive method can contain diagnostic systemsthat rely on bioluminescence for visualizing tissues in situ. Thesesystems can further include compositions containing substrates that areconverted by the TKTL1 enzymatic activity. In particular these systemscan include a composition that contains a bioluminescence generatingreaction. Administration of the compositions results in production oflight by targeted tissues that permits the detection and localization ofcells or tissues e.g. for surgical removal.

For performing the inventive method in course of a molecular imaging, inparticular a radiographic imaging of tissue, it is proposed to use aradio-opaque imaging agent that in one embodiment accumulatesintracellular in tissue in proportion to its functional, orphysiological, activity. In one embodiment, the imaging agent is a cellmembrane-permeable, radio-opaque, selective substrate or high affinityligand for TKTL1.

The present invention therefore also relates to labeled TKTL1 substratesand the use of same as imaging agents, for example as positron emissiontomography (PET) imaging agents or magnetic resonance tomography (MRT)imaging agents for the noninvasive detection and localization of cellsand tissues with an enhanced or reduced TKTL1 enzymatic activity.

A very suitable labelled substrate for use as imaging agents for PET is¹⁸F-labeled TKTL1. The invention further relates to methods ofsynthesizing labelled substrates and to compositions comprising suchanalogues.

In course of the experiments leading to the present invention it wasfurther found that mutations of TKTL1 (e.g. during evolution of mammals,a deletion of an exon encoding 38 amino acids happened in TKTL1) notonly result in the lowering of substrate specificity but, in addition,in a lower affinity for thiamine. Thus, a reduced thiamine level leadsto an increased failure of some of the TKTL1 proteins resulting indamages being due to decreased TKTL1 activity. These pathologicalchanges can be avoided or at least corrected by activation of thismetabolic pathway. Determination of thiamine affinity of TKTL1, oramount of TKTL1 or activity of TKTL1 can be exploited to identifyindividuals who should be treated with thiamine or thiamine-derivateswhich a better bioavailability (e.g. benfotiamine).

Such individuals may be, for example diabetes patients having diabetesassociated phenomenons like retinopathy, (cardiac autonomic) neuropathy,or damaging of endothelial cells.

In consequence the present invention further relates to a method forcontrolling the mam-aGF in an mammalian subject (patient) in need ofsuch controlling, wherein the controlling comprises administering of aneffective amount of at least one inhibitor or activator of the activityor concentration of the enzyme TKTL1.

With other words: The present invention further relates to the use of aninhibitor or activator of the activity or concentration of the enzymeTKTL1 for manufacturing a pharmaceutical composition for the inhibitionor activation of the mam-aGF, i.e. for therapeutic treatment orcontrolling of diseases associated with an decreased or enhancedmam-aGF.

This technical teaching is based on the scientific finding (in course ofthe novel and surprising discovery of the mam-aGF), that a furtherimportant property of the TKTL1 enzyme is its appropriateness as atarget molecule for an inhibitor or activator of the mam-aGF.

The treatment of disorders associated with overexpression of TKTL1 genemay comprise any method suitable for the reduction of the activity ofTKTL1 polypeptide in an individual or in cells of an individual. Thesemethods may comprise a reduction of the activity of TKTL1 polypeptide bymeans of reduction of gene expression or by means of reduction ofenzymatic activity. Examples may comprise the administration ofantisense constructs, of ribozymes, of enzyme inhibitors, theadministration of antagonists of cofactors of TKTL1 polypeptides, suchas e.g. antithiamine compounds or the reduced administration ofessential cofactors for the enzymatic activity (e.g. thiamine).

A preferred therapy of disorders associated with the overexpression ofTKTL1 gene comprises administration of antithiamine compounds or thereduction of thiamine uptake for individuals showing disorderscharacterized by overexpression of TKTL1 gene.

In consequence the present invention also comprises a pharmaceuticalcomposition comprising an effective amount of an inhibitor or activatorof the activity or concentration of the enzyme TKTL1 and apharmaceutically acceptable carrier.

A preferred embodiment of such a pharmaceutical composition comprises aTKTL1 inhibitor elected from the group consisting of oxythiamine,benfooxytiamine (=oxybenfotiamin), hydroxypyruvate, pyruvate,p-hydroxyphenylpyruvate, pyrithiamin, amprolium, 2-methylthiamin,2-methoxy-p-benzochinon (2-MBQ) and 2,6-dimethoxy-p-benzochinon(2,6-DMBQ), genistein, and flavonols as e.g. quercetin, catechins,nitrilosides, anthocyanins; or derivatives thereof.

A preferred embodiment of such a pharmaceutical composition with TKTL1inhibitor effect comprises one or more Amprolium derivatives having thechemical structure (structural formular):

and/or at least one Flavonol having the chemical structure (structuralformular):

Another preferred embodiment of a pharmaceutical composition with TKTL1inhibitor effect comprises one or more thiamin and/or benfotiaminderivatives having the chemical structure (structural formular)

for thiamine derivatives and (b):

for benfotiamine derivatives.

One preferred inhibitory benfotiamine derivative is oxybenfotiamine(=benfooxytiamin) having the chemical structure (structural formular):

A preferred embodiment of a pharmaceutical composition with TKTL1activator effect comprises thiamine and/or benfotiamine and/orfunctionally equivalent, i.e. activating, derivates thereof.

The activating thiamine derivatives preferably have the chemicalstructure (structural formula):

And the activating benfotiamin derivates preferably have the chemicalstructure (structural formula):

Derivatives of the above listed activators or inhibitors can begenerated by substituting or adding one or more of the following groups:

linear and branched (C₁-C₁₂) aliphatic alkyl groups, substituted with atleast one group chosen from OH, NH₂, SH, CN, CF₃, halogen, CONHR⁵,COOR⁵, OR⁵, SR⁵, SiOR⁵, NHR⁵, aliphatic (C₃-C₆) rings, and aromatic(C₃-C₆) rings, wherein R⁵ is chosen from linear and branched (C₁-C₄)alkyl groups,aryl groups,natural polymers, synthetic polymers, and copolymers, said polymers andcopolymers carrying at least two groups chosen from: hydroxyl,carboxylate, primary amine, secondary amine, tertiary amine, thiol, andaldehyde;a hydrogen atom, a halogen atom, CF₃, OH, OCF₃, COOH, R⁷, OR₇, andOCOR⁷, wherein R⁷ is chosen from linear and branched (C₁-C₄) aliphaticalkyl groups;a monohalogenated and polyhalogenated linear and branched (C₁-C₄) alkylgroups, and from aryl groups, wherein the aryl groups are optionallysubstituted with at least one group chosen from OH, NH₂, SH, CN, CF₃,halogen, COOH, CONHR⁸, COOR⁸, OR⁸, SR⁸, and NHR⁸, wherein R⁸ is chosenfrom linear and branched (C₁-C₁₂) alkyl radicals; andfrom linear and branched (C₁-C₄) alkyl groups, and a CF₃ group.

The TKTL1 inhibitors or activators may also be realized in form of oneor more nucleic acid molecules, recombinant vectors, polypeptides,antisense RNA sequence, ribozymes and/or antibodies.

Therefore another pharmaceutical composition of the present inventionwhich is suitable for the prevention or treatment of a diseaseassociated with an abnormal cellular localization of the mutated TKTL1protein, aggregation status and/or dimerization status (compared to acontrol contraception) is characterised in that it comprises aneffective amount of a nucleic acid molecule, a recombinant vector, apolypeptide, an antisense RNA sequence, a ribozyme or an antibody.

This pharmaceutical composition may for example contain DNA that codesfor a functional TKTL1. The DNA may be administered in a way that allowsthe polypeptides to be generated in situ. Suitable expression systemsare known to those skilled in the art. Transgenic mammalian cells may beused for delivery and/or expression of the nucleic acids. Theappropriate methods are known to those of skill in the art.

Alternatively, the pharmaceutical compositions may comprise one or morepolypeptides. The polypeptides incorporated into pharmaceuticalcompositions may be the TKTL1 polypeptides in combination with one ormore other known polypeptides such as for example enzymes, antibodies,regulatory factors, such as cyclins, cyclin-dependent kinases or CKIs,or toxins.

When blood sugar levels rise, some key kinds of cell—including nervecells (neurons) and the cells that make up the fine blood cells of theretina of the eye and the filtering units (glomeruli) of the kidney—arealso flooded with glucose. The resulting high sugar levels within thesecells cause a logjam in the normal cellular metabolism of glucose. Thisbacklog results glucose associated cell damages and in a buildup withinthe cell of super-reactive glucose-metabolic intermediates known astriosephosphates leading to advanced glycation endproducts (AGE). Andonce that happens, the excess glucose and triosephosphates attack thesurrounding proteins, lipids, and DNA within the cell.

An enhancement of the mam-aGF by activating the TKTL1 could be acorrective and preventive in that situation. By the mam-aGF pathwayglucose is degraded to nontoxic compounds like fatty acids thereforeavoiding advanced glycation endproduct damages.

Therefore, the invention relates to a method for the treatment ofglucose and triosephosphate associated cell damages and prevention ofAGE-associated cell damages in a patient in need of such treatmentcomprising administering an effective amount of at least one TKTL1activator to said patient.

With other words: Therefore the invention also relates to the use of atleast one TKTL1 activator for manufacturing a pharmaceutical compositionfor the treatment and prevention of AGE-associated cell damages in apatient.

A defection/disturbance of the mam-aGF pathway is not generallyassociated with a serious disease but sometimes merely associate withdisturbances of the physical stage which indeed are subjectiveunpleasant, but do not need a medical treatment. Furthermore the mam-aGFpathway can be controlled not only by activating or inhibiting the TKTL1enzyme but also by limiting the substrate (glucose) for this metabolicpathway. Therefore the present invention further provides nutrientcompositions/dietary supplements containing a low glucose/carbohydratecontent, a high oil/fat content and a moderate protein content andfurther comprising an effective amount of an inhibitor or activator ofthe activity or concentration of the enzyme TKTL1 and a pharmaceuticallyacceptable carrier.

A preferred embodiment of such a nutrient composition/dietary supplementcomprises a TKTL1 inhibitor elected from the group consisting ofoxythiamine, benfooxytiamine, hydroxypyruvate, pyruvate,p-hydroxyphenylpyruvate, pyrithiamin, amprolium, 2-methylthiamin,2-methoxy-p-benzochinon (2-MBQ) and 2,6-dimethoxy-p-benzochinon(2,6-DMBQ), genistein, and flavonols as e.g. quercetin, catechins,nitrilosides, anthocyanins; or derivatives thereof

An other preferred embodiment of such a nutrient composition/dietarysupplement comprises a TKTL1 activator, especially thiamin orbenfotiamine or one or more derivatives thereof. Said derivatives arepreferably characterised by the above-mentioned chemical structure(structural formulas).

The present invention also relates to the use of a compound for thetreatment of a disease associated with an enhanced or decreased level oractivity, an abnormal cellular localization of the TKTL1 protein,aggregation status and/or dimerization status compared to a control,wherein said compound is a compound identified by a method comprisingthe following steps:

(a) contacting a TKTL1 polypeptide as defined in the preceding claims ora cell expressing said polypeptide in the presence of components capableof providing a detectable signal in response to a biological activity,preferably transketolase activity; and

(b) detecting presence or absence of a signal or increase of the signalgenerated from said biological activity, wherein the absence, decreaseor increase of the signal is indicative for a putative drug.

The drug candidate may be a single compound or a plurality of compounds.The term “plurality of compounds” is to be understood as a plurality ofsubstances which may or may not be identical. Said compound or pluralityof compounds may be chemically synthesized or microbiologically producedand/or comprised in, for example, samples, e.g., cell extracts from,e.g., plants, animals or microorganisms. Furthermore, said compound(s)may be known in the art but hitherto not known to be capable ofsuppressing or activating TKTL1 polypeptides. The reaction mixture maybe a cell free extract or may comprise a cell or tissue culture.Suitable set ups are known to the person skilled in the art. Theplurality of compounds may be, e.g., added to the reaction mixture,culture medium, injected into a cell or otherwise applied to thetransgenic animal. The cell or tissue that may be employed in the methodof the invention preferably is a host cell, mammalian cell or non-humantransgenic animal of the invention described in the embodimentshereinbefore.

If a sample containing a compound or a plurality of compounds isidentified in the method of the invention, then it is either possible toisolate the compound from the original sample identified as containingthe compound capable of suppressing or activating TKTL1, or one canfurther subdivide the original sample, for example, if it consists of aplurality of different compounds, so as to reduce the number ofdifferent substances per sample and repeat the method with thesubdivisions of the original sample. Depending on the complexity of thesamples, the steps described above can be performed several times,preferably until the sample identified according to the present methodonly comprises a limited number of or only one substance(s). Preferablysaid sample comprises substances of similar chemical and/or physicalproperties, and most preferably said substances are identical.

The compounds which can be tested and identified may be peptides,proteins, nucleic acids, antibodies, small organic compounds, hormones,peptidomimetics, PNAs or the like. These compounds may also serve aslead compounds for the development of analog compounds. The analogsshould have a stabilized electronic configuration and molecularconformation that allows key functional groups to be presented to theTKTL1 in substantially the same way as the lead compound. In particular,the analog compounds have spatial electronic properties which arecomparable to the binding region, but can be smaller molecules than thelead compound, frequently having a molecular weight below about 2 kD andpreferably below about 1 kD. Identification of analog compounds can beperformed through use of techniques such as self-consistent field (SCF)analysis, configuration interaction (CI) analysis, and normal modedynamics analysis. Computer programs for implementing these techniquesare available; e.g., Rein, Computer-Assisted Modeling of Receptor-LigandInteractions (Alan Liss, New York, 1989).

As known up to now the novel discovered mam-aGF is regularly (normally,natively) present in healthy tissues like retina, endothelial cells,nervous tissue and testis.

In the course of the experiments leading to the present invention itbecame obvious that in several tumor tissues the mam-aGF proceeds withhigh turn-over. In these tumor cells an overexpressing of the TKTL1enzyme was detected and likewise large amounts of lactate leading to amatrix degradation. These findings confirm the studies of Warburg, whodetected that not only in the absence of oxygene but also in thepresence of oxygen tumor tissue degrades glucose to lactate, and thatthere is a correlation between the degree of aerobic fermentativeglucose degradation (aerobic glycolysis), lactate production andmalignancy. By transcript and protein based analysis of the three TKTfamily members it could be demonstrated, that the TKTL1 gene is thetransketolase gene/protein which is overexpressed in tumors.

Therefore the present invention also comprises a method for thetreatment of cancer associated with an overexpression of TKTL1 enzyme inthe tumor cells in a patient in need of such treatment comprisingadministering an effective amount of at least one TKTL1 inhibitor tosaid patient.

With other words: Therefore the invention also relates to the use of atleast one TKTL1 inhibitor for manufacturing a pharmaceutical compositionfor the treatment of cancer associated with an overexpression of TKTL1enzyme in the tumor cells.

Since it is known that large amounts of lactate lead to a matrixdegradation and that this facilitates tissue remodeling and woundhealing, the data and knowledges obtained in the course of theexperiments leading to the present invention result in a further part ofthe invention, namely in a method for treatment (or influencing theprocesses) of tissue remodeling, wound healing etc in a patient in needof such treatment comprising administering an effective amount of atleast one TKTL1 inhibitor or TKTL1 activator to said patient.

With other words: The invention also relates to the use of at least oneTKTL1 inhibitor or TKTL1 activator for manufacturing a pharmaceuticalcomposition for enhancing or reducing the processes of tissueremodeling, wound healing etc.

An embodiment of such pharmaceutical composition comprising at least oneTKTL1 inhibitor is intended to be applied in restenosis in heart valvesto prevent proliferation of endothelial cells.

Since many years transketolase proteins and transketolase enzymeactivities have been related to neurodegenerative diseases likeWernicke-Korsakoff syndrome, Alzheimer disease patients, etc. However,the question, how this relation looks like remains open until completionof the present invention.

In the course of the experiments leading to the present invention it wasfound, that both, patients with Alzheimer disease and patients withWernicke-Korsakoff syndrome, have reduced TKTL1 enzyme activities andTKTL1 protein variants with different isoelectric points or smallersize. In patients with Wernicke-Korsakoff syndrome it could bedemonstrate that their cells do harbor TKTL1 protein isoforms with areduced affinity for thiamin. Since glycation (e.g. glucose iscovalently bound to the proteins—the chemical reaction is known asSchiff's base reaction) is one of the processes involved inamyloidogenesis and protein plaque generation leading toneurodegenerative diseases (for example: the fibrils present inAlzheimer's disease patients share several properties common to glycatedproteins and glycation causes the structural transition from folded,soluble form to beta-fibrils) reduced TKTL1 enzyme activities results inan enhancement of glycation and finally in enhanced amyloidogenesis andprotein plaque generation.

Due to these facts the present invention also comprises a method fortreatment of neurodegenerative diseases like Wernicke-Korsakoffsyndrome, Alzheimer disease etc. by inhibiting glycation via activationof the mam-aGF in a patient in need of such treatment comprisingadministering an effective amount of at least one activator of TKTL1enzyme activity to said patient (e.g. thiamin or benfotiamineapplication in food).

Furthermore, enhancing the TKTL1 activity and thereby the mam-aGF canprevent the development of Alzheimer's disease in individuals with apredisposition due to TKTL1. Thus, the present invention furthercomprises a method to determine individuals with TKTL1 predisposingvariants to identify individuals eligible for a preventive TKTL1therapy.

In this context it should be mentioned that unwanted apoptosis in cellslike neurons can be reduced or blocked by treatment of TKTL1 withactivating compounds identified by the methods according to theinvention.

For patients with diabetes mellitus it has been shown recently thatbenfotiamine treatment of such patients effects the blockade of threemajor pathways of hyperglycemic damage and prevents diabeticretinopathy. Now, in the course of the experiments leading to thepresent invention the inventors were able to identify the TKTL1 enzymeas the target of that benfotiamin treatment in those tissues(endothelial cells, retina, and nerves) in which patients with diabetesget cell damage due to AGE (advanced glycation end-product) formation orcell death.

AGE is also caused by an excess of glucose and triosephosphates withincells. An enhancement of the mam-aGF by activating the TKTL1 wouldresult in a degradation of glucose and triosephosphates.

Due to these facts the present invention also comprises a method for thetreatment of AGE in patients, especially diabetes patients, in need ofsuch treatment, comprising administering an effective amount of at leastone activator of TKTL1 enzyme activity to said patient, preferablybenfotiamine.

In the course of the experiments leading to the present invention it wasfurther found that reducing the TKTL1 activity can cause growthretardation and preferential reduction of adipose tissue. By inhibitionof TKTL1 in adipose tissue obesity can be treated.

Diseases like systemic lupus erythematosus (SLE), rheumatoid arthritis,multiple sclerosis (MS), fibromyalgia, crohn's disease, irritable bowelsyndrome (IBS) have steadily risen for the past 80 years. TargetingTKTL1 lead to a significant decrease in levels of auto-antibodies inpatients with autoimmune diseases.

The present invention will be explained by means of the followingexamples and figure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. (A) Quantification of TKTL1 transcripts in gastric and lungadenocarcinoma samples and their corresponding normal tissues. N—normalsample; T—tumor sample; M—marker, 100 bp and 200 bp fragments are shown.

FIG. 2: (A) Expression pattern of the human TKTL1 gene on northern blotsof poly(A)⁺ mRNA from different human adult tissues analysed with aTKTL1 cDNA probe.

-   -   (B) Expression of TKTL1 protein isoforms in five tumor cell        lines derived from four different tumor entities.    -   (C) TKTL1 full length protein expressed in E. coli.

FIG. 3: Determination of transketolase activity of native (A) andrecombinant (B) TKTL1 protein.

FIG. 4: TKTL1 protein expression in

-   -   A-B: normal corpus tissue of gastric carcinoma patient 1;    -   C-F: tumor tissue of gastric carcinoma patient 1;    -   G-I: normal antrum tissue of gastric carcinoma patient 2,    -   J-N: gastric carcinoma cells of patient 2,    -   O,P: poorly differentiated gastric carcinoma;    -   Q: colon carcinoma;    -   R: superficial bladder carcinoma;    -   S,T: invasive poorly differentiated bladder carcinoma.    -   Magnification: G×50; C,H, and J×100; A,B,D,E,I,K,M,O, and S×200;        F,L,N,P,Q,R and T×400.

FIG. 5: TKTL1 staining of noninvasive and invasive bladder carcinoma.

FIG. 6-8: Expression of TKTL1 and phosphorylated Akt (ph-Akt) inparaffin embedded sections of

-   -   A-C: normal, papillary (PTC), follicular (FTC), and        undifferentiated (UTC) thyroid cancer; D: normal and NSLC        tissues, E: colon cancer; F: normal bladder; G: prostate        carcinomas,    -   AEC=red staining; Counterstaining with haematoxylin=blue        staining,    -   Yellow arrowheads indicate nuclear ph-AKT staining

FIG. 9-10: Expression of TKTL1 in endothelial cells.

FIG. 11-12: Expression of TKTL1 in neuronal cells.

FIG. 13-14: schematic drawing of the mam-aGF pathway.

FIG. 15: 2-dimensional (2D)-gel electrophoresis of a multi-proteincomplex harboring TKTL1 protein isoforms (arrow A), DNaseX, and GAPDH.

FIG. 16: 2D-gel electrophoresis of high molecular weight TKTL1 proteinisoforms (arrow) identified by immunostaining.

FIG. 17: ELISA determination (A) of TKTL1 protein isoforms and (B) oftransketolase activity of isolated TKTL1 protein. Values obtained

-   -   (A)A1-A5: from leukocytes from healthy persons,    -   (A)A6-A10: from fibroblasts from healthy persons,    -   (A)B1-B5: from serum from healthy persons,    -   (A)B6-B10: from brain cells from healthy persons,    -   (A)A11-A12: without probe material (background level),    -   (A)B11-B12: without probe material (background level),    -   (A)C1-C3: from leukocytes from AD patients,    -   (A)C4-C6: from fibroblast from AD patients,    -   (A)C7-C9: from serum from AD patients,    -   (A)C10-C12: from brain cells from AD patients.    -   (A)D1-D3: from leukocytes from Morbus Parkinson patients,    -   (A)D4-D6: from fibroblast from Morbus Parkinson patients,    -   (A)D7-D9: from serum from Morbus Parkinson patients,    -   (A)D10-D12: from brain cells from Morbus Parkinson patients,    -   (A)E1-E3: from leukocytes from Huntington disease patients.    -   (A)E4-E6: from fibroblast from Huntington disease patients.    -   (A)E7-E9: from serum from Huntington disease patients,    -   (A)E10-E12: from brain cells from Huntington disease patients,    -   (A)F1-F3: from leukocytes from SLE patients,    -   (A)F4-F6: from fibroblast from SLE patients.    -   (A)F7-F9: from serum from SLE patients.    -   (A)F10-F12: from kidney cells from SLE disease patients,    -   (A)G1-G3: from leukocytes from Morbus Parkinson patients.    -   (A)G4-G6: from fibroblast from Morbus Parkinson patients.    -   (A)G7-G9: from serum from Morbus Parkinson patients,    -   (A)G10-G12: from kidney cells from Morbus Parkinson disease        patients,    -   (A)H1-H3: from leukocytes from diabetes type II Morbus Parkinson        patients,    -   (A)H4-H6: from fibroblast from diabetes type II patients,    -   (A)H7-H9: from serum from diabetes type II patients,    -   (A)H10-H12: from kidney cells from diabetes type II disease        patients,    -   (B)A1-A5: from leukocytes from healthy persons,    -   (B)A6-A10: from fibroblasts from healthy persons,    -   (B)B1-B5: from serum from healthy persons,    -   (B)B6-B10: from brain cells from healthy persons,    -   (B)A11-A12: without probe material (background level),    -   (B)B11-B12: without probe material (background level),    -   (B)C1-C3: from healthy persons,    -   (B)C4-C6: from neuronal cells from healthy persons,    -   (B)C7-C9: from kidney cells from healthy persons,    -   (B)C10-C12: from colon cells from healthy persons,    -   (B)D1-D3: from (B)leukocytes from AD patients,    -   (B)D4-D6: from fibroblast from AD patients,    -   (B)D7-D9: from serum from AD patients,    -   (B)D10-D12: from brain cells from AD patients,    -   (B)E1-E3: from leukocytes from Morbus Parkinson patients,    -   (B)E4-E6: from fibroblast from Morbus Parkinson patients,    -   (B)E7-E9: from serum from Morbus Parkinson patients,    -   (B)E10-E12: from brain cells from Morbus Parkinson patients,    -   (B)F1-F3: from leukocytes from SLE patients,    -   (B)F4-F6: from fibroblast from SLE patients,    -   (B)F7-F9: from serum from SLE patients,    -   (B)F10-F12: from kidney cells from SLE disease patients,    -   (B)G4-G6: from fibroblast from multiple sclerosis patients,    -   (B)G7-G9: from serum from multiple sclerosis patients,    -   (B)G10-G12: from kidney cells from multiple sclerosis patients,    -   (B)H1-H3: from leukocytes from diabetes type II patients,    -   (B)H4-H6: from fibroblast from diabetes type II patients,    -   (B)H7-H9: from serum from diabetes type II patients,    -   (B)H10-H12: from kidney cells from diabetes type II disease        patients.

GENERAL INFORMATION CONCERNING THE EXAMPLES

Origin and Cultivation of Cells

The lung carcinoma cell line A549, the breast carcinoma cell line MCF7,the liver carcinoma cell line HepG2, and the colon carcinoma cell linesHCT116 and HT29 were obtained from ATCC. Cells were grown in RPMI 1640or DMEM supplemented with 10% FCS, penicillin and streptomycin(Invitrogen) at 37° C. with 5% CO₂.

Northern Blot Analysis

A DNA probe from the 3′ untranslated region (residue 1627 to 2368) ofthe TKTL1 transcript (acc. no. X91817) was labelled with[[alpha]-³²P]dATP and [[alpha]-³²P]dCTP (3000 Ci/mmol) in a randomprimed reaction (Feinberg and Vogelstein, 1983). Hybridization wascarried out in 0.5 M sodium phosphate, 7% SDS, 0.2% bovine serumalbumin, 0.2% PEG 6000, 0.05% polyvinylpyrrolidone 360000, 0.05% Ficoll70000 and 0.5% dextran sulphate at 65° C. overnight. Non-specificallybound probe was removed by washing at 65° C. in 40 mM sodium phosphate,pH 7.2, 1% SDS for 60 min. Filters were exposed to X-ray film (Kodak)for 1-5 days. A multiple human adult tissue poly(A)⁺ RNA northern blotwas purchased from BD Biosciences Clontech.

Western Blot Analysis

For Western blot analysis, cells were lysed in lysis buffer (50 mMTris-HCl pH 7.5, 150 mM sodium chloride, 1% NP40, 0.5% sodiumdeoxycholate, 0.1% SDS, 0.02% sodium azide, 1 mM phenylmethylsulfonylfluoride). Aliquots of 50 μg of soluble protein was loaded into eachwell, electrophoresed on 12.5% SDS-polyacrylamide gels, and transferredto polyvinylidene difluoride membranes (Millipore). For detection ofTKTL1-proteins the HRP-coupled JFC12T10 MAb was used in a finalconcentration of 1 μg/ml. The MAb was visualized with an ECL Westernblot detection system (Amersham Pharmacia Biotech).

Enzyme-Linked Immunosorbent Assay (ELISA)

TKTL1 protein affinity-purified from cell lines was determined usingcommon standard ELISA techniques. Three different affinity-purifiedmouse IgG monoclonal anti-TKTL1 antibodies (5 μg/ml) were used forcoating of ELISA plates. Horseradish peroxidase conjugated anti-TKTL1antibody JFC12T10 was used at 5 μg/ml as the secondary reagent. Boundproteins in the multi-protein complex were affinity-purified from celllines using antibodies directed TKTL1, DNaseX, ph-Akt, GAPDH. Binding tocertain proteins was assessed by ELISA technique, using e.g. thecombination of TKTL1 and GAPDH antibodies; and the combination of TKTL1and DNaseX antibodies; and the combination of ph-Akt and DNaseXantibodies.

2D-Analysis of Multi-Protein Complexes

The samples were analysed by high resolution 2D gel electrophoresis (8×7cm). 2.5 μg of protein was applied to two 2D gels for each sample and 2Dgel was stained by silver and the proteins of the second one weretransferred to PVDF membranes by semidry electroblotting forimmunostaining.

Example 1 Expression Pattern of the Human TKTL1 Gene on Northern Blotsof Poly(A)+mRNA from Different Human Adult Tissues Analysed with a TKTL1cDNA Probe

Expression pattern of the human TKTL1 gene was analysed with a TKTL1cDNA probe on Northern blots of poly(A)⁺ mRNA from different human adulttissues. The results are shown in FIG. 2 (A). Four transcripts of 1.4,1.9, 2.5, and 2.7 kb are detectable. Whereas the main transcript in mosttissues is 2.5 kb in size, in heart the small transcript of 1.4 isabundant and the 2.5 and 2.7 kb transcripts are missing. Transcriptsizes are indicated in kb.

Example 2 Isolation and Purification of the TKTL1 Full Length Protein

The TKTL1 full length protein was expressed in E. coli and was isolatedby affinity purification through the N-terminal His-tag. One fig ofaffinity purified TKTL1 protein was loaded onto a 4-20%-gradient SDS geland stained with Coomassie. Proteins different in size were detected.The largest protein (66 kDa) represents the N-terminal His-tagged fulllength TKTL1 protein, whereas smaller TKTL1 proteins are likely due toC-terminal proteolytic cleavage already present prior to isolationprocedure. Note that the migration of the recombinant 66 kDa His-taggedTKTL1-full length protein indicates a size of 75 kDa. Sizes of theprotein marker are indicated in kDa.

Example 3 Determining the Level of TKTL1 Gene Expression in Tissues byMeasuring the TKTL1 mRNA Levels

Dissections of biopsies can be semi-quantitatively analysed for the mRNAlevel of TKTL1 gene in an in-situ staining reaction. The stainingreaction is performed as follows:

The tissue dissections are incubated in ascending ethanol concentrationsup to 100% ethanol. After evaporation of the alcohol the dissections areboiled in 10 mM citrate buffer (pH 6.0) for pre-treatment of the tissue.The hybridization mixture is prepared by mixing 50 μl of ready to usehybridization buffer (DAKO A/S, Glostrup, Danmark) with about 5-10 pmolof the probes. The probes are fluorescein-labelled oligonucleotides ofthe following sequence: TCTCATCACAAGCAGCACAGGAC

Example 4 Determination of Transketolase Activity of Native (A) andRecombinant (B) TKTL1 Protein

The two-substrate and one-substrate reaction of native (A) andrecombinant (B) TKTL1 protein was determined by the production of NADHas measured by gain in absorbance at 340 nm. Xylulose-5-phosphate (X5P)and ribose-5-phosphate (R5P) were used to determine the two-substratereaction, whereas X5P alone was used for the one-substrate reaction. InFIG. 3 one representative of three independent enzymatic assays leadingto similar results is shown.

Example 5 Determination of TKTL1 Isoforms by ELISA

The combination of TKTL1 antibody JFC6T8 and JFC5T3 determines a TKTL1protein isoform specifically present in patients with neurodegenerativediseases. The antibodies JFC6T8 and JFC5T3 were coupled to an ELISAplate, and incubated with samples from healthy persons and patients.After removing unspecific bound material, enzymatic activity wasdetermined as described above. High enzymatic activities were obtainedin samples of healthy persons. The individual results are listed in FIG.17 (A)

Example 6 Identification of High Molecular Weight TKTL1 Protein Isoformsby 2D-Gel Electrophoresis

High molecular weight TKTL1 protein isoforms from a patient with aneurodegenerative disease (AD) were isolated analyzed by 2D-gelelectrophoresis and identified by immunostaining. The results are shownin FIG. 16.

Example 7 Multi-Protein Complex Harboring TKTL1, DNaseX, and GAPDH

The multi-protein complex harboring TKTL1, DNaseX, and GAPDH wasaffinity-purified from human chronic myelogenous leukemia K562 cellsusing TKTL1 antibody JFC12T10 coupled to carbo-link. A 2-dimensional(2D)-gel electrophoresis of that multi-protein complex was carried out.TKTL1 protein isoforms (arrow A) and other proteins present in thecomplex were identified by immunostaining and sequence determination.The results are shown in FIG. 15.

Example 8 Transketolase Activity of TKTL1 Isolated from Healthy andPatient Derived Specimens, Determined with ELISA

TKTL1 antibody JFC3T9 was coupled to an ELISA plate, and incubated withsamples from healthy persons and patients. After removing unspecificbound material, enzymatic activity was determined as described above.High enzymatic activities were obtained in samples of healthy persons.The individual results are listed in FIG. 17 (B).

Example 9 Assays for Detection of Compounds for Enhancing or Reducingthe TKTL1 Enzyme Activity

Since the TKTL1 protein isoforms represent moonlighting proteins,different assays for identifying active small compounds have to beapplied. Assays for detection of compounds for enhancing or reducing theTKTL1 enzyme activity can be performed by the recombinant proteinisoforms or by native protein isoforms isolated from human cells.

(A) Providing Recombinant TKTL1 Protein Isoforms

This can be realized by expression of recombinant TKTL1 protein isoformsin E. coli. The TKTL1 open reading frame (MADAE . . . CMLLN) of cDNAsequence (acc. no. BC025382) was cloned into the pDEST17 vector(Invitrogen). Bacterial expression was performed in the E. coli strainBL21-AI (Invitrogen), and expression was induced with 0.2% arabinose at21° C. for 4 h. Crude cell lysate was prepared in lysis buffer (20 mMTris[pH7.5], 5 mM imidazole, 5 mM beta-mercaptoethanol, 500 mM NaCl, and1% Triton X-100) by freezing (dry ice, 10 min) and thawing (37° C., 5min) 3 times. Soluble protein fractions were obtained by centrifugationof the cell lysate at 12.000×g for 30 min at 4° C. His₆-TKTL1 proteinwas purified with Ni-NTA resins (Qiagen) according to the manufacturer'sinstructions with elution buffer containing 200 mM imidazole. Imidazoleand salt were subsequently removed by dialysis against 0.1 M Tris (pH7.5). The purified enzyme was stored at −20° C. in 40% glycerol and 0.1%dithiothreitol (DTT).

(B) Providing Native Protein Isoforms Isolated from Human Cells

Native TKTL1 proteins and protein complexes harboring TKTL1, bothharvested from human cell lines, must be purified, for example viaaffinity-purification. This can be realized as following:

10 mg of MAb JFC12T10 was coupled to 2 ml carbo-link according to themanufacturer's instructions (carbo-link; Pierce). Cells were grown inserum free media (ISF-1, InVivo BioTech Services GmbH). Aftercentrifugation, the pellet of 2.2×10⁹ cells was resolved in 50 ml PBScontaining protease inhibitors cocktail (Roche). A cell lysis wasperformed using a french press, followed by a centrifugation at50.000×g. The supernatant was filtered (0.2 μm) and binding ofsupernatant to affinity material was performed over night at 4° C.(batch modus). After transfer to a column, a wash procedure wasperformed with 150 mM PBS buffer pH 7.4. For elution of column attachedproteins 100 mM Glycin-HCl pH 2.0 was used. Two proteins peaks, detectedusing a UV 280 nm-based detection system, have been collected andneutralized with Tris pH 7.4.

Enzymatic tests can be performed with the recombinant or the native,affinity purified TKTL1 protein.

(C) Detection of Suitable Compounds by Determining the Two-SubstrateTransketolase Reaction.

(C-1) The transketolase (two-substrate) activity of TKTL1 was measuredby a coupled enzyme assay at 25° C. Reactions were started by additionof recombinant and native TKTL1 protein (a) in the presence of the testcompound and (b) in the absence of the test compound and determinedspectrophotometrically by the rate of reduction of NAD⁺ in the followingreaction sequence: xylulose-5-phosphate (X5P) and ribose-5-phosphate(R5P)>(TKTL1 activity)>glyceraldehyde-3-phosphate,sedoheptulose-7-phosphate>(glyceraldehyde-3-phosphate dehydrogenaseactivity [GAPDH])>NAD⁺→NADH+H⁺, 1,3-phosphoglycerate.

Transketolase two-substrate activity (a) in the presence of the testcompound and (b) in the absence of the test compound was determined inthe following reaction (final concentrations): 4 mM X5P, 4 mM R5P, 500μM NAD⁺, 2 mM MgCl₂, 200 μM thiamine PP, 5 μg recombinant TKTL1 protein,or 4 μg native TKTL1 protein, 3 U GAPDH, 0.15 mol/l Tris buffer pH 7.4in a reaction volume of 1 ml. Transketolase one-substrate activity wasdetermined by omitting R5P, using X5P solely as substrate. GAPDH wasobtained from Sigma.

(C-2) Transketolase activity (a) in the presence of the test compoundand (b) in the absence of the test compound can be measured by using aconventional enzyme-linked method under conditions in which couplingenzymes are not limiting. Reactions are initiated by the addition oftransketolase protein to an otherwise complete reaction mix of 100mmol/L Tris-HCl (pH 7.5), 10 mmol/L ribose 5-phosphate, 2 mmol/Lxylulose 5-phosphate, 1.2 mmol/L MgCl₂, 0,1 mmol/L NADH, 2000 U/Lglycerol-3-phosphate dehydrogenase and triose phosphate isomerase.Reactions are conducted at 37° C. The oxidation of NADH, which isdirectly proportional to transketolase activity, is followed bymonitoring the decrease in absorbance at 340 nm.

(C-3) Substrates (in variable concentrations) can be tested as possibledonors if erythrose-4-phosphate (1 mM) as acceptor is used. In such areaction fructose-6-phosphate will be build. With the enzymesglucose-6-phosphate-dehydrogenase and 6-phosphoglucose-isomerase,fructose-6-phosphate will be oxidized to 6-phosphogluconolactone,leading to the generation of NADPH.

(C-4) Formaldehyde (variable concentrations) as acceptor can be usedleading to dihydroxyacetone. The following reaction ofglycerin-dehydrogenase builds glycerin, concomitant with an oxidation ofNADH.

(D) Determining the One-Substrate Transketolase Reaction

(D-1) Via Oxidation of NADH:

Transketolase activity (a) in the presence of the test compound and (b)in the absence of the test compound is measured by using a conventionalenzyme-linked method under conditions in which coupling enzymes are notlimiting. Reactions are initiated by the addition of transketolaseprotein (a) together with the test compound and (b) in the absence ofthe test compound to an otherwise complete reaction mix of 100 mmol/LTris-HCl (pH 7.5), 5 mmol/L xylulose 5 phosphate, 1.2 mmol/L MgCl₂, 3mmol/L phosphate, 0.1 mmol/L NADH, 2000 U/L glycerol-3-phosphatedehydrogenase and triose phosphate isomerase. Reactions are conducted at37° C. The oxidation of NADH, which is directly proportional totransketolase activity, is followed by monitoring the decrease inabsorbance at 340.

(D-2) Via Reduction of NAD:

Transketolase activity (a) in the presence of the test compound and (b)in the absence of the test compound is measured by using a conventionalenzyme-linked method under conditions in which coupling enzymes are notlimiting. Reactions are initiated by the addition of transketolaseprotein (a) together with the test compound and (b) in the absence ofthe test compound to an otherwise complete reaction mix of 100 mmol/LTris-HCl (pH 7.5), 5 mmol/L xylulose 5 phosphate, 3 mmol/L phosphate,1.2 mmol/L MgCl₂, 0.1 mmol/L NAD, 2000 U/L glyceraldehyde-3-phosphatedehydrogenase. Reactions are conducted at 37° C. The reduction of NAD,which is directly proportional to ketolase activity, is followed bymonitoring the increase at 340 nm. In addition, generation ofacetyl-phosphate can be measured.

(E) Determining Transketolase Reaction with Further Substrates

Transketolase activity (a) in the presence of the test compound and (b)in the absence of the test compound is measured by using a conventionalenzyme-linked method under conditions in which coupling enzymes are notlimiting. Reactions are initiated by the addition of transketolaseprotein (a) together with the test compound and (b) in the absence ofthe test compound to an otherwise complete reaction mix of 100 mmol/LTris-Cl (pH 7.5), 5 mmol/L acetaldehyde, 5 mmol/L pyruvate, 1.2 mmol/LMgCl₂. The reaction leads to 3-hydroxybutanon (acetoin) and CO₂.Reactions are conducted at 37° C. Transketolase activity is measured byHPLC-chromatography.

Further substrates can be:

(a) Formaldehyde and pyruvate leading to hydroxyacetone and CO₂.

(b) Glycerinaldehyde and pyruvate leading to 1-desoxyxylulose and CO₂.

(F) In Vivo Assays for Identification of TKTL1 Inhibitors Based onLactate Production

Cell lines, which can be tested are, e.g., glioblastoma cell line LN18,colon cancer cell line HT29, breast cancer cell line MCF7. Cell lineshave to be grown in media containing 2 mg/ml glucose (a) in the presenceof the test compound and (b) in the absence of the test compound.

Glucose consumption and lactate production has to be determined for 5days. Every day the glucose and lactate content in the media is tested.As an additional control, glioblastoma cell line LN229 could be used,which does not show high glucose consumption and a high lactateproduction rate.

Example 10 Screening Methods for Drug Candidates

(A) Assays for Single Compound Testing

Cell lines (e.g., as described above) should be grown with and withoutthe compound to be tested. As synthetic test compounds, for example,thiamin, oxythiamin, p-hydroxyphenylpyruvate, pyrithiamin, amprolium,2-methylthiamin, benfooxytiamine, benfotiamine, 2-methoxy-p-benzochinon(2-MBQ) and 2,6-dimethoxy-p-benzochinon (2,6-DMBQ), genistein, andflavonols as e.g. quercetin, catechins, nitrilosides and anthocyanins orderivatives of them can be used.

Derivatives of the above listed compounds can be generated bysubstituting or adding one or more of the following groups:

linear and branched (C₁-C₁₂) aliphatic alkyl groups, substituted with atleast one group chosen from OH, NH₂, SH, CN, CF₃, halogen, CONHR⁵,COOR⁵, OR⁵, SR⁵, SiOR⁵, NHR⁵, aliphatic (C₃-C₆) rings, and aromatic(C₃-C₆) rings, wherein R⁵ is chosen from linear and branched (C₁-C₄)alkyl groups, aryl groups,natural polymers, synthetic polymers, and copolymers, said polymers andcopolymers carrying at least two groups chosen from: hydroxyl,carboxylate, primary amine, secondary amine, tertiary amine, thiol, andaldehyde;a hydrogen atom, a halogen atom, CF₃, OH, OCF₃, COOH, R⁷, OR₇, andOCOR⁷, wherein R⁷ is chosen from linear and branched (C₁-C₄) aliphaticalkyl groups;a monohalogenated and polyhalogenated linear and branched (C₁-C₄) alkylgroups, and from aryl groups, wherein the aryl groups are optionallysubstituted with at least one group chosen from OH, NH₂, SH, CN, CF₃,halogen, COOH, CONHR⁸, COOR⁸, OR⁸, SR⁸, and NHR⁸, wherein R⁸ is chosenfrom linear and branched (C₁-C₁₂) alkyl radicals; andfrom linear and branched (C₁-C₄) alkyl groups, and a CF₃ group.

Natural products, or extracts or fractions thereof can also be used toidentify compounds for activating or inhibiting TKTL1 enzymaticactivity, e.g., fermented wheat germ extract AVEMAR or apple extracts.Substrates or substrate-analogues specific for TKTL1 can be used toaccelerate or inhibit TKTL1 enzymatic activity. Reactions specific forthe TKTL1 protein isoforms can be exploited to inhibit or activate TKTL1enzyme activities. Compounds which inhibit the TKTL1 enzymatic activitycan be used to prevent obesity.

Thus, compounds can be identified which will lead to a reduced glucoseconsumption or lactate production. Such compounds are, e.g., useful forreducing obesity, for reducing or inhibiting spermatogenesis, lactateproduction in sperms (leading to a matrix degradation in uterus) thuscan be applied as contraceptives.

Moreover, compounds can be identified which will lead to an enhancedglucose consumption or lactate production. Such compounds can be used,e.g., for accelerating wound healing and bone repair, for reducing andnormalizing blood glucose levels in diabetes mellitus patients, forpreventing or reducing pathological alterations in small and largevessels in diabetes mellitus patients, for reducing retinopathies orneuropathies in diabetes mellitus patients and for inhibiting orpreventing neurodegenerative diseases like Alzheimer disease,Wernicke-Korsakoff syndrome, Huntington disease, and Morbus Parkinson.

(B) Assays for Determining Compounds Influencing the Protein-ProteinInteractions of the Mutated TKTL1 Protein Isoforms

Protein-protein interactions play a role both in regulating enzymaticactivity and in signal transduction pathways that regulate cellularfunction. The number of small molecules protein-protein interactioninhibitors (SMPIIs) is growing rapidly. Living cells are continuouslyexposed to a variety of signals from their micro- and macro-environment.Many of these signals are detected by receptors present on the cellsurface, and are then processed and transduced by intracellularsignalling cascades. Because the ultimate site of action in a signallingcascade is often far from the cell surface, an inherent feature ofintracellular signalling pathways is the requirement that proteinstranslocate from one position to another within the cell. Thesetranslocations, and thus cell signalling and response, depend criticallyon protein-protein interactions that mediate protein translocationthrough the intracellular space.

As an example of a typical signal transduction pathway involving proteintranslocation, the signalling and protein translocation steps involvedin the cellular response of the phosphatidylinositol 3 kinase (PI3K)pathway to a growth factor such as insulin is depicted. This pathwayinfluences and is influenced by TKTL1.

1. Insulin binds to and activates its receptor at the cell surface. Uponactivation, the receptor recruits adaptor proteins and activatesintracellular signalling molecules including PI3K.

2. Activated PI3K increases the plasma membrane concentration of thelipid phosphatidylinositol 3,4,5-triphosphate (PIP3).

3. PIP3 in the plasma membrane provides docking sites for proteinkinases including Akt1/PKBa and PDK1; Akt is activated by PDK1 only whenboth are docked at the membrane. This translocation step is an absoluterequirement for Akt activation.

4. Once activated by PDK1 at the plasma membrane, Akt is free to diffuseback into the cell interior, where it can phosphorylate substrate suchas the transcription factor Forkhead (FKHR, FOXOA1).

5. Unphosphorylated FKHR normally resides in the nucleus, where itmodulates genes involved in cell cycle arrest and apoptosis. However,once phosphorylated by Akt1, FKHR translocates to the cytoplasm, whereit can no longer modulate target genes.

Protein-protein interactions and translocations are involved at each ofthese steps, notably for Akt1 and Forkhead. Thus, a signal initiated bythe binding of insulin to a cell surface receptor modulates thetranscription of genes involved in cellular growth and survival via asequential cascade of protein translocation events. The therapeuticrelevance of this becomes clear when one considers that alteredsignalling responses are often key distinguishing features between cellsin normal and diseased tissues.

(C) Assays for Small-Molecule Protein-Protein Interaction Inhibitors

Historically, large peptides and natural products have been consideredthe primary compound classes capable of modulating protein-proteininteractions. However, there is growing evidence in the literature andfrom screening initiatives to suggest that small molecules can alsomodulate the interactions responsible for protein-protein complexes.These compounds may act either directly—via inhibition at theprotein-protein interface—or indirectly—via binding to an allostericsite and induction of a conformational change of the target protein oran associated molecule.

Traditional small molecule drug discovery focuses primarily on theactivity of compounds against purified targets, such as binding tocell-surface receptors or inhibition of the catalytic activity ofenzymes. While these approaches have led to the development of a largenumber of useful drugs, they clearly have limitations. Because of thecomplex network environment in which intracellular signalling occurs, itis advantageous to screen compounds in living cells to reproduce thepathway and network context in which the drug will eventually have toact. When employed as part of a pathway screening strategy, cell-basedtranslocation assays offer an opportunity to discover and progressentirely new classes of compounds that act primarily by modulatingprotein interactions.

Cell-based assays that monitor the intracellular behaviour of targetmolecules, rather than binding or catalytic activity of purifiedproteins, can now be used in high-throughput screens to discover andprofile SMPPIIs.

Known transketolase (TKT) genes encode a single protein with enzymaticactivity, whereas TKTL1 transcripts and proteins different in size havebeen detected. Furthermore, part of the TKTL1 protein(s) is present inthe nucleus of cells. Therefore the one gene/one protein/one functionrelationship is wrong for the TKTL1 gene. Known transketolases arehomodimers of two full length proteins harbouring all typical invarianttransketolase amino acid residues. The transketolase-like gene encodedTKTL1 protein isoforms build TKTL1 homo/heterodimers and TKT/TKTL1 (andTKTL2/TKTL1) heterodimers. The expression of TKTL1 protein isoforms—evenan enzymatically non-active—influences the enzymatic activity of a TKTprotein as part of a TKT/TKTL1 heterodimer. The same is also true forTKTL2/TKTL1 heterodimers. A molecular switch and a proton wiresynchronizes the active sites in TKT/TKTL1 heterodimers and TKTL1/TKTL1homo- and heterodimers. Another type of protein interaction is presentas TKTL1 protein isoforms are part of a multi-protein complex. TKTL1proteins are bound to transketolase unrelated proteins like GAPDH,DNaseX (DNA acc. No. X90392; protein acc. No. CAA62037),(phosphorylated-)Akt, histone, histone acetylase, actin binding protein,and amyloid precursor protein (APP). The presence or binding of eachmember of the multi-protein complex changes. The changes are influencedby the translocation of cytoplasmic localized proteins to the nucleus.Once arrived in the nucleus, the former cytosolic proteins do exertsfunctions different to the function within the cytoplasm. We havedetected a translocation of DNaseX from the cytoplasm to the nucleus inapoptotic and tumor cells. We have also detected a translocation ofph-Akt from the cytoplasm to the nucleus in tumor cells (FIG. 6-8). Atranslocation of GAPDH has been detected in apoptotic neuronal cells.The release from cytoplasmic binding sites or the new synthesis ofproteins, which are directly translocated into the nucleus, leads tomulti-protein complexes which are inducing apotosis. This apoptosis isthe basis for the death of cells, e.g. neurons in brains of patientswith neurodegenerative diseases. In tumor cells the sucide moleculeDNaseX is present in the nucleus, (but exerts no DNase activity,) whichwould lead to apoptosis and cell death of the tumor cells. Instead,binding to this multi-protein complex leads to inactivation of DNaseX intumor cells. Therefore apoptosis is blocked. In neurodegenerativediseases DNaseX, GAPDH and TKTL1 lead to apoptosis of cells, cells whichshould not die. The unwanted apoptosis lead to the severe effects.

Bound proteins in the multi-protein complex were affinity-purified fromcell lines using antibodies directed against TKTL1, DNaseX, ph-Akt, andGAPDH. Binding to certain proteins was assessed by ELISA technique,using e.g. the combination of TKTL1 and GAPDH antibodies; thecombination of TKTL1 and DNaseX antibodies; the combination of TKTL1 andph-Akt antibodies; the combination of TKTL1 and TKT antibodies; thecombination of TKTL1 and TKTL2 antibodies.

(D) In Vivo High-Throughput Screen to Discover and Profile SMPPIIs forInfluencing Protein-Protein Interactions of TKTL1 Protein Isoforms

SMPPIIs can be identified which influence the generation of TKTL1homo/heterodimers and TKT/TKTL1 heterodimers and the interaction toother proteins of the multi-protein complex. SMPPIIs can be identifiedwhich influence the generation of TKTL1 protein interactions with suchother proteins e.g. DNaseX, GAPDH or amyloid beta peptide (A beta).SMPPIIs can be identified which influence the generation of TKTL1protein aggregates.

SMPPIIs can be identified which influence the protein-proteininteraction with other proteins and the following generation of proteinaggregates e.g. GAPDH or amyloid beta peptide (A beta). SMPPIIs can beidentified which influence the translocation of TKTL1 protein isoformse.g. translocation from cytoplasm to nucleus.

The altered substrate specificity and reaction modus of the TKTL1 enzymecan be used for the destruction of cells or tissues with an enhancedTKTL1 enzyme activity. Application of a nontoxic substrate can beapplied to patients with enhanced TKTL1 enzyme activity. Cells with anenhanced expression of TKTL1, harbor a gene product (TKTL1 enzyme) whichtargets the cells for selective killing. Those cells, which show anenhanced TKTL1 enzymatic activity, convert the nontoxic substrate into atoxic drug by rendering the cells sensitive to a nontoxic prodrug or achemotherapeutic agent, thereby eliminating unwanted cells. Thisstrategy of killing unwanted cells can be e.g. applied for epithelialcell (head and neck, oesophagus, gastric, colon and rectum andurothelial cells) by administering nontoxic prodrugs e.g. in food.

(E) Mutations within the TKTL1 Gene Have Been Detected Leading to TKTL1Protein Isoforms with Different Isoelectric Properties and ReducedAffinities for Thiamine:

A test can be performed identifying mutations within the TKTL1 gene byDNA-based methods. A test can be performed by isolating TKTL1 proteinisoforms using a monoclonal antibody specific for the TKTL1 protein(s).The antibody could be attached to microtiter plates. Serum or othersamples could be analyzed and the TKTL1 protein isoform can be isolatedform these specimens. A standardized enzymatic transketolase test couldbe performed allowing the determination of transketolase activity orKm-values for thiamine. Using this procedure, individuals with reducedTKTL1 activities could be identified prior to the beginning of thedisease e.g. diabetes mellitus, Wernicke-Korsakoff syndrome, Huntingtondisease. Those patients should be treated with a TKTL1 activatorcompound.

An in vivo assay with cells can be performed for screening smallcompound inhibiting the translocation to the nucleus or the aggregationof TKTL1 protein within the nucleus, as monitored e.g. by (in vivo)immunohistochemical methods. Cells can be analysed for the presence ofhigh molecular weight complexes harbouring TKTL1 or the presence ofprotein complexes with reduced solubility. The above mentioned in vivoassays can also be performed using a TKTL1-GFP fusion protein.

Example 11 Controlling of the mam-aGF Via Nutrition Based Therapy

A further embodiment of the invention relates to a novel therapeuticapproach which is based on the expression of TKTL1 and its concomitantsugar metabolism. Besides the inhibition of TKTL1 enzymatic activity bysmall compounds or inhibitory substrates, TKTL1 enzymatic activity canalso be inhibited by limited substrate availability through applicationof a targeted nutrition. The targeted nutrition based therapy orprevention consists of a test for the determination of TKTL1 enzymaticactivity in tumors or non malignant cells/tissues followed by a specificnutrition.

The basic nutrition consists of a selected fatty acids composition,preferably in an amount of 55 to 65% (w/w); a selected carbohydratecomposition, preferably, in an amount of 5 to 15% (w/w) with,preferably, less than 2% (w/w) glucose (or starch) content, preferably,mainly comprising fructose, oligofructose, galactose, oligogalactose; aselected protein (aminoacid) composition, preferably, in an amount of 10to 25% (w/w) with, preferably, more than 40% (w/w) (lysine, leucine),and, preferably, more than 30% (w/w) (isoleucine, phenylalanine,threonine, tryptophan, tyrosine); tocotrienol and electron acceptors orcombination thereof:

A preferred embodiment consist:

-   -   a) 62% of a combination of fatty acids (see Table 1);    -   b) 12% carbohydrates with less than 2% glucose (or starch)        content, mainly consisting of fructose, oligofructose,        galactose, oligogalactose;    -   c) 18% proteins with more than 40% (lysine, leucine), and more        than 30% (isoleucine, phenylalanine, threonine, tryptophan,        tyrosine)    -   d) Tocotrienol (e.g. gamma-tocotrienol)    -   e) at least one electron acceptor, such as, for example,        parabenzoquinones, benzoquinones, hydroxyquinones and derivates        thereof.

The basic nutrition in combination with a pharmaceutically acceptablecarrier and thiamin or thiamin derivates (etc. benfotiamine) which areactivating the TKTL1 enzymatic activity will be applied to prevent ortreat neurodegenerative diseases, diabetes, diabetes complications,metabolic syndrome, macro- and microvascular damages, aging, retinalcell damage, central, inflammation of endothelial cells, and peripheralneuronal cell damage, because in normal (not malignant) cells like, forexample, retina cells, central and peripheral neurons, and endothelialcells, the TKTL1 activity protects from damaging effects of insufficientsugar metabolism leading to AGE or radical formation.

For cancer treatment the daily basic nutrition has to be adjusted,preferably, to a maximal total amount of 0.2 mg thiamine. This can bedone by selection of nutrition with low thiamine level, by thiaminasetreatment of nutrition or by heating/boiling of nutrition. The basicnutrition with a pharmaceutically acceptable carrier and low levels ofthiamine or the basic nutrition with low levels of thiamine supplementedwith inhibitory thiamine analogs (etc. oxythiamin, oxybenfotiamine) isadministered to cancer patients, if a high TKTL1-activity and/ortranscript/protein concentration in their tumors or metastases isdetected. This nutritional approach leads to an inhibition of TKTL1enzymatic, thereby reducing glucose metabolism and inhibiting tumorproliferation.

TABLE 1 Example of a fatty acid mixture in weight %: caprylic acid (C8)46.6 capric acid (C10) 28.2 linoleic acid (ω6-C18:2) 3.6 SDA (ω3-C18:4)0.2 ETA (ω3-C20:4) 0.3 EPA (ω3-C20:5) 5.7 DPA (ω3-C22:5) 0.9 DHA(ω3-C22:6) 4.9 other 9.6 total MCFA's 74.8 total n-3 PUFA's 12.0 totalother 13.2 DHA:EPA 0.86 n-3:n-6 3.1 MCFA = Medium chain fatty acids,i.e. fatty acids having 8-14 carbon atoms), PUFA = Polyunsaturated FattyAcids, i.e. fatty acids having more than one double bond)

Example 12 Detecting of the TKTL1-Protein-Level in Cancer Tissue and inNormal (Healthy) Tissue of Thyroidea, Lung and Colon

Five μm thick human cancer and normal paraffin sections of thyroidtissue, lung tissue and colon tissue were analyzed byimmunohistochemistry. Dewaxed sections were heated for antigen unmaskingin 10 mM sodium citrate (pH 6.0) for 1 minute at 450 W followed by 5minutes at 100 W. After rinse in dH₂O, inhibition of endogenousperoxidase was performed with 5 min incubation with 3%-H₂O₂. Then,sections were exposed to biotin blocking system (DAKO) for 10 min toblock endogenous avidin-biotin. After two washes in Tris/saline buffer(TBS), slides were incubated with 1% goat serum for 30 min to blockunspecific staining. Successively, sections were exposed to mouseanti-TKTL1 (clone JFC12T10; mouse IgG2_(b)) antibody (25 μg/ml) oranti-Ser473 phospho-Akt (587F11; mouse IgG2_(b); Cell SignalingTechnology) overnight at 4° C. Then slides were washed in TBS andincubated with biotinilated anti-mouse immunoglobulins for 30 min atroom temperature and treated with streptavidin-peroxidase (DAKO).Staining was revealed using 3-amino-9-ethylcarbazole (AEC) substrate.Nuclei counterstaining was performed using aqueous haematoxylin.

The results of that immunohistochemical staining are shown in FIGS. 6, 7and 8. For each cancer type one representative of three independentexperiments is shown. TKTL1 and phosphorylated Akt are highly expressedin thyroid cancer tissue. Non small lung cancer (NSLC) and coloncarcinomas express high levels of TKTL1 and phosphorylated Akt.

Example 13 Detecting of the TKTL1-Protein-Level in Tumors of GastricCarcinoma Patients, Colon Carcinoma Patient and Noninvasive and InvasiveBladder Carcinoma Patients

The TKTL1 protein expression in tumors of three gastric carcinomapatients (FIG. 4 A-P), one colon carcinoma patient (FIG. 4 Q), onenoninvasive bladder carcinoma patient (FIG. 4 R) and one invasivebladder carcinoma patient (FIG. 4 S-T) was determined and compared withcorresponding normal tissue.

TkTL1 protein determination was carried out by help of a monoclonalanti-TKTL1 antibody. The anti-TKTL1 antibody was revealed bydiaminobenzidine tetrahydrochloride (DAB; brown staining).Counterstaining was performed with haematoxylin (blue staining).

The specimens of gastric carcinoma patient 1 reveal strong cytoplasmicexpression of TKTL1 in tumor tissue but no expression in the surroundingstroma cells (FIG. 4 C-F). Note the heterogenous expression in tumorcells (FIG. 4 E-F) The corresponding normal tissue shows no expressionof TKTL1 (FIG. 4 A-B).

The specimens of gastric carcinoma patient 2 reveal strong cytoplasmicexpression within tumor cells (FIG. 4 J-N) and heterogenous expressionin tumor cells (FIG. 4 L). The corresponding normal antrum tissue showsno expression of TKTL1 (FIG. 4 G-I).

The specimens of gastric carcinoma patient 3 reveal nuclear expressionin a poorly differentiated gastric carcinoma (FIG. 4 O-P).

The specimens of colon carcinoma patient reveals cytoplasmic staining(FIG. 4 Q).

The specimens of the patient with superficial bladder carcinoma revealsno expression of TKTL1 (FIG. 4 R).

The specimens of the patient with an invasive poorly differentiatedbladder carcinoma reveals strong cytoplasmic expression (FIG. 4 S-T).

A comparison of noninvasive and invasive bladder carcinoma tissue isshown in FIG. 5. The non-invasive bladder carcinoma tissue shows non oronly few staining which indicates no expression of TKTL1 while theinvasive bladder carcinoma tissue shows strong staining that indicatesstrong expression of TKTL1.

Example 14 Expression of TKTL1 Protein Isoforms in Five Tumor Cell LinesDerived from Four Different Tumor Entities

The expression of TKTL1 protein isoforms in five tumor cell linesderived from four different tumor entities were detected using a MAbspecifically detecting TKTL1 protein isoforms and not reacting withother transketolase family members. The results are shown in FIG. 2 (B).Each cell line do show a unique expression pattern of TKTL1 proteinisoforms. The molecular weight standard is indicated in kDa.

Example 15 Expression of TKTL1 and Phosphorylated Akt (ph-Akt) in Cancerand Normal Tissue

Immunohistochemical analysis of TKTL1 or ph-Akt was carried out onparaffin-embedded sections of normal, papillary (PTC), follicular (FTC),and undifferentiated (UTC) thyroid cancer (FIG. 6 A-C), of normal andNSLC tissues (FIG. 7 D), of colon cancer (FIG. 7 E) and of normal orbladder and prostate cancer (FIG. 8 F-G) with Anti-TKTL1 or anti-ph-Akt.Anti-TKTL1 or anti-ph-Akt was revealed by 3-amino-9-ethylcarbazole (AEC;red staining). Counterstaining was performed with haematoxylin (bluestaining). Negative controls were performed using isotype matched IgG.

TKTL1 is mainly localized within the cytoplasm, but a nuclear stainingcan also be identified in a subset of tumors. Phosphorylated Akt islocalized within the cytoplasm and/or the nucleus.

Example 16 Detecting of the TKTL1-Level in Patients with GastricCarcinoma, Patients with Colon Carcinoma, Patients with NoninvasiveBladder Carcinoma and Patients with Invasive Bladder Carcinoma

Three μm thick paraffin sections were heated for antigen unmasking in 10mM sodium citrate (pH 6.0) for 5 minutes at 900 W, for 5 min at 900 W indH₂0, and for 5 min in 10 mM sodium citrate (pH 6.0) at 900 W. After awash in phosphate/saline buffer (PBS), inhibition of endogenousperoxidase was performed as above described. Then, sections were exposed15 min to biotin-avidin blocking buffer (Vector Laboratories). Blockingof unspecific staining was performed with goat serum as described above.Primary antibodies were visualized with avidin-biotinylated horseradishperoxidase complex (ABC) and diaminobenzidine tetrahydrochloride (DAB)(Elite kit; Vector Laboratories), and counterstained with Mayer'shaematoxylin.

The results of the immunohistochemical stainings are shown in FIGS. 4,5,and 9-16.

Example 17 Quantification of TKTL1 Transcripts in Gastric and LungAdenocarcinoma Samples and their Corresponding Normal Tissues

15 μl of the real-time PCR reaction was loaded onto a 3%-agarose gel tovisualize the 150 bp TKTL1 amplification product. Expression differencesbetween tumor and corresponding normal tissue have been calculated onbasis of the real-time data and are shown as fold-induction in tumorsample relative to the corresponding normal sample. (B) Real-timetranscript quantification of TKT, TKTL1, TKTL2, and β-actin gene in alung adenocarcinoma and corresponding normal sample. The highestexpression level is observed for the β-actin. Within the transketolasegene family, the TKT gene shows the highest expression level. The TKTL1and TKTL2 expression level in normal lung is low compared to that of TKTand β-actin. In contrast to this, the expression level of TKTL1 in lungadenocarcinoma is 60-fold higher than in the corresponding normaltissue.

Example 18 Diagnosis of Neurodegenerative Diseases

Fibroblast cell lines, foreskin fibroblasts or leukocytes from healthysubjects and patient with Alzheimer's disease, or otherneurodegenerative diseases were analyzed for TKTL1 abnormalities bymeans of ELISA, electrofocusing gel analysis, 2D-gel electrophoresis andimmunostaining. The ELISA experiments were performed by different ELISAapproaches. One type of ELISA represents a typical ELISA, where catchingor detecting antibody is directed against a certain protein. The othertype of ELISA used consisted of an antibody directed against one proteinand an antibody against another protein. An example for type 1 ELISA isthe combination of TKTL1 antibody JFC12T10 and TKTL1 antibody JFC10T9.JFC12T10 detects an epitope of the TKTL1 protein, and does not crossreact with TKT or TKTL2. JFC10T9 detects another epitope of TKTL1. Usingthe ELISA JFC12T10/JFC10T9 TKTL1 protein can be detected and measured.An example for Type 2 ELISA is antibody JFC12T10 directed against TKTL1and antibody JFC11D8 directed against DNaseX. Using this ELISA theprotein interaction of TKTL1 and DNaseX can be determined. Both types ofELISA reactions were performed with samples from healthy persons andpatients. One type of sample consisted of body fluids like serum and wasdirectly analysed for the presence of proteins and protein interactions.Another type of analysis was performed using an antibody (e.g. JFC12T10or JFC11D8) coupled to carbo-link. Using affinity purificationprocedures we isolated multi-protein complexes of cells (derived fromcell culture or native tissues). The multi-protein complexes wereanalysed by electrofocusing or 2D-gel electrophoresis followed byimmunostaining or determination of enzymatic activity (e.g.transketolase two- or one-substrate reaction; DNase test, GAPDHactivity). Using these assays, protein isoforms could be identifiedspecifically present in patients with neurodegenerative diseases likeAD. In patients with neurodegenerative disorders like AD patients, TKTL1variants have been detected with high alkaline pI, lower two- orone-substrate reaction, and lower thiamin affinity. Additionally, usingstandard PAGE smaller protein isoforms and a higher amount of smallerprotein in comparison to full length TKTL1 were detected in intact cellsor cell extracts from those patients compared to healthy persons.Furthermore reduced two- or one-substrate reaction of TKTL1, or lowerthiamin affinity of TKTL1 has been observed in healthy persons whichlater on (month and years later) showed neurodegenerative disease likeAD. The observed TKTL1 variants lead to reduced sugar metabolism incells. These reduced sugar metabolism lead to enhanced AGE formation andAGE formation lead to high molecular protein aggregates and cell death.This unwanted cell death of cells necessary for proper brain function,is an important cause for these neurodegenerative diseases. To identifyindividuals which do have TKTL1 variants with a reduced two- orone-substrate reaction or lower thiamin affinity, TKTL1 antibodies wereestablished, which can be used to isolate TKTL1 proteins from samples tobe tested (e.g. JFC12T10). Those samples can be body fluids (e.g. serum)or cell samples (e.g. proteins of fibroblasts or leukocytes). TKTL1antibodies coupled to ELISA plates catch the TKTL1 proteins and afterwashing away TKT and TKTL2 proteins, the (trans-)ketolase two- or onesubstrate reaction can be enzymatically determined in a coupledenzymatic reaction by e.g. building the reduced NADH (the enzymaticassays are described above). Similarly the enzymatic reaction wasperformed at different concentrations of thiamin. By reducing thethiamine level in the assay TKTL1 variants were identified in patientswith neurodegenerative diseases with a reduced affinity for thiamine.Using this approach of ELISA and enzymatic analysis, TKTL1 variants canbe identified which predispose to neurodegenerative diseases at atimepoint before signs of neurodegenerative diseases are present. Thiscan be exploited for a prevention of neurodegenerative diseases e.g. byapplication of better soluble thiamine derivates like benfotiamine or adiet with reduced levels or certain types of sugars (e.g. glucose). Inaddition to the identification of TKTL1 variants with a reduced two- orone-substrate reaction or lower thiamin affinity, TKTL1 variants with areduced solubility or TKTL1 variants present in high molecular weightcomplexes were identified in neurodegenerative disease patients like AD.The inventors established TKTL1 specific antibodies specificallyreacting with TKTL1 variants present in high molecular weight complexesin nuclei of patients with neurodegenerative disease patients like AD(JFC7T4). Using ELISA reactions or immunohistochemical stainings, thosedisease specific TKTL1 variants can be identified in body fluids (e.g.serum), or in tissue samples (e.g. leukocytes, fibroblasts, biopsies).Furthermore in combination with antibodies directed against otherproteins present in those multi-protein complexes, ELISA can beperformed detecting the presence of protein interactions. Such an type 2ELISA consisting of TKTL1 antibody JFC8T7 and DNaseX antibody JFC7D4identified a protein interaction of TKTL1 and DNaseX, specific for cellsgoing into apoptosis. Another type 2 ELISA consisting of TKTL1 antibodyJFC8T7 and GAPDH antibody JFC3G6 identified a protein interaction ofTKTL1 and GAPDH, specific for cells going into apoptosis. The presenceof these protein complexes can be exploited for the detection andtherapy of neurodegenerative diseases. The identification of suchprotein interactions between TKTL1 and other proteins like GAPDH,DNaseX, and ph-Akt can be exploited for the isolation of antiapoptoticcompounds. Those compounds can be used as pharmaceutical agents for thetreatment of neurodegenerative diseases. Compounds specifically bindingto TKTL1 can be identified by affinity labeling, and e.g. by means ofBIAcore technology. Antiapoptotic effect can be detected using reducedprogrammed cell death (visualized by e.g. apoptotic ladder, caspase-3,annexin). TKTL1 and GAPDH are tightly bound to each other. The TKTL1(trans-)ketolase reaction cleaving sugars like X5P leads to theproduction of GAP. As GAPDH is tightly bound to TKTL1, the produced GAPis directly used from GAPDH which lead to the production1,3-phosphoglycerate concomitant with the reduction of NAD⁺ to NADH+H⁺.For the isolation of small compounds inhibiting TKTL1 and GAPDHinteractions different NAD⁺ concentrations should be used since thebinding of some compounds is depend on the concentration of NAD⁺.Another type of protein interaction was detected using antibody JFC12T10as sole antibody. If the TKTL1 protein is present as a single protein,no ELISA reaction should work if just one antibody is used as catchingand detecting antibody. In case of TKTL1 antibody JFC12T10 can be usedas catching and detecting antibody. Therefore, using this antibodyprotein interactions of TKTL1 and another TKTL1 protein can be detected.As some of the TKTL1 protein isoforms miss N-terminal protein sequencesdimers consisting of TKTL1 and TKTL1 can be discriminated into homo- andhetero TKTL1-dimers. Some of the dimers consists of full length TKTL1protein bound to another full length TKTL1 protein (TKTL1 homodimer).Some of the dimers consists of a full length TKTL1 protein and a smallerTKTL1 isoform, where the N-terminus is missing. The discrimination canbe performed using TKTL1 antibodies located at different sites withinthe TKTL1 protein. E.g. N-terminal located TKTL1 antibodies can be usedwith C-terminal located antibodies and the result of this ELISA can becompared with an ELISA using only C-terminally located antibodies. Theratio between those two ELISA results can be used for the identificationof patients and for the identification of healthy persons which willlater on get a TKTL1-associated disease. (See also FIG. 11-12)

Example 19 Expression of TKTL1 in Endothelial Cells

The majority of normal tissues and cells do show no expression of TKTL1.In the retina, in endothelial cells and in neuronal cells an expressionof TKTL1 is present. Retina, endothelial cells and neuronal cells getdamaged by high glucose levels. As shown in FIG. 9 and FIG. 10 TKTL1protein is expressed in the nucleus and/or the cytoplasm of endothelialcells (and retina, and neuronal cells; not shown).

1. A method for qualitatively and quantitatively detecting an extent ofuse of mammalian aerobic glucose fermentation metabolic pathway(mam-aGF) and a normal process flow of said pathway in a mammalianindividual, wherein transketolase-like 1 (TKTL1) protein is used asindicator and target molecule or a TKTL1 nucleic acid or a fragmentthereof is used as a target molecule or indicator, said methodcomprising: taking a biological sample of said individual, determiningan activity and/or concentration, and/or cellular localization and/oraggregation status and/or dimerization status of the TKTL1 protein orthe TKTL1 nucleic acid within said sample of said individual and withina control sample, comparing determined data obtained from said sample ofsaid individual with data obtained from the control sample, wherein (i)an enhanced or decreased level of activity and/or concentration of theTKTL1 protein or of the TKTL1 nucleic acid in said sample of theindividual compared to the control sample provides an indication of anenhancement or decrease in said extent of use of mammalian aerobicglucose fermentation metabolic pathway, and (ii) an abnormal cellularlocalization and/or an abnormal aggregation status and/or an altereddimerization of the TKTL1 protein in said sample of the individualcompared to the control sample provides an indication of an abnormalprocess flow of said mammalian aerobic glucose fermentation metabolicpathway.
 2. The method of claim 1, wherein a disease associated with anenhanced or decreased and/or abnormal mammalian aerobic glucosefermentation metabolic pathway is detected and monitored.
 3. The methodaccording to claim 1 or 2, wherein the method is used in the course ofan in vivo or in vitro molecular imaging method.
 4. The method accordingto claim 1 or 2, wherein the biological sample is a tissue sample, abiopsy, a body fluid, a secretion, a smear, serum, urine, semen, stool,bile, a liquid containing cells, lysed cells, cell debris, peptides ornucleic acids.
 5. The method according to claim 4, wherein thedetermination is carried out with the TKTL1 protein as the targetmolecule.
 6. The method according to claim 5, wherein the determinationis carried out by using a molecule that specifically binds to the TKTL1protein.
 7. The method according to claim 6, wherein said molecule is anantibody directed to TKTL1 or a fragment of such anti-TKTL1-antibody ora peptidomimetic comprising an antigen binding epitope, or amini-antibody.
 8. The method according to claim 4, wherein thedetermination is carried out with a TKTL1 gene or TKTL1 mRNA as thetarget molecule.
 9. The method according to claim 8, wherein at leastone nucleic acid probe capable of hybridizing to the TKTL1 gene or aTKTL1 mRNA is used for the determination.
 10. The method according toclaim 8 or 9, wherein a chimeric nucleic acid comprising a TKTL1 nucleicacid is used for the determination.
 11. The method of claim 1, whereinsaid the mammalian aerobic glucose fermentation metabolic pathway isinhibited or activated.
 12. A method for qualitative and quantitativelydetecting an extent of use of the mammalian aerobic glucose fermentationmetabolic pathway (mam-aGF) and a normal process flow of said pathway ina mammalian individual, wherein transketolase-like 1 (TKTL1) protein isused as indicator and target molecule or a TKTL1 nucleic acid is used asa target molecule or indicator, said method comprising: taking abiological sample of said individual, determining an activity and/orconcentration, and/or cellular localization and/or aggregation statusand/or dimerization status of the TKTL1 protein or the TKTL1 nucleicacid within said sample of said individual, comparing the determineddata obtained from said sample of said individual with reference dataobtained by determining the activity and/or concentration, and/orcellular localization and/or aggregation status and/or dimerizationstatus of the TKTL1 protein or the TKTL1 nucleic acid within a controlsample, wherein (i) an enhanced or decreased level of activity and/orconcentration of the TKTL1 protein or of the TKTL1 nucleic acid in saidsample of said individual compared to the control sample provides anindication of an enhancement or decrease in said extent of use of themammalian aerobic glucose fermentation metabolic pathway, and (ii) anabnormal cellular localization and/or an abnormal aggregation statusand/or an altered dimerization of the TKTL1 protein in said sample ofsaid individual compared to the control sample provides an indication ofan abnormal mammalian aerobic glucose fermentation metabolic pathway.